Optical instrument comprising multi-notch beam splitter

ABSTRACT

An instrument is provided that can monitor nucleic acid sequence amplification reactions, for example, PCR amplification of DNA and DNA fragments. The instrument includes a multi-notch filter disposed along one or both of an excitation beam path and an emission beam path. Methods are also provided for monitoring nucleic acid sequence amplifications using an instrument that includes a multi-notch filter disposed along a beam path.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a continuation of U.S. patent application Ser. No.12/321,830, filed Jan. 26, 2009, which is a continuation of U.S. patentapplication Ser. No. 10/456,196, filed Jun. 6, 2003, (now U.S. Pat. No.7,498,164), which is a continuation-in-part of U.S. patent applicationSer. No. 10/216,620, filed Aug. 9, 2002, (now U.S. Pat. No. 7,008,789)which is a continuation of U.S. patent application Ser. No. 09/700,536,filed Nov. 29, 2001, (now U.S. Pat. No. 6,818,437) which is a NationalPhase Application Under 35 U.S.C. §371 of PCT International ApplicationNo. PCT/US99/11088, filed May 17, 1999, which claims benefit of U.S.Provisional Patent Application No. 60/085,765, filed May 16, 1998, andalso claims benefit of U.S. Provisional Patent Application No.60/092,784, filed Jul. 14, 1998. This application is also related toU.S. patent application Ser. No. 10/370,846, filed Feb. 20, 2003. All ofthe above-identified applications are incorporated herein in theirentireties by reference.

FIELD

This teachings relates to biochemical analyses, and particularly toquantitative monitoring of nucleic acid sequence amplification reactionprocesses.

BACKGROUND

Quantitative measurements can be made on the amount of DNA productionduring a polymerase chain reaction (PCR) process, to providemeasurements of the starting amount and the amount produced.Measurements and computation techniques are taught in U.S. Pat. No.5,766,889 (Atwood), as well as in the article “Kinetic PCR Analysis:Real-time Monitoring of DNA Amplification Reactions” by Russel Higuchi,et al., Bio/Technology vol. 11, pp. 1026-1030 (September 1993), and inthe article “Product Differentiation by Analysis of DNA Melting Curvesduring the Polymerase Chain Reaction” by Kirk M. Ririe, et al.,Analytical Biochemistry vol. 245, pp. 154-160 (1997), which areincorporated herein in their entirety by reference.

There is a need for greater precision during monitoring and measuringPCR and other nucleic acid amplification techniques. Previousinstruments that allow real time acquisition and analysis of PCR datacan be very basic devices without the required dynamic range, can bewithout built-in calibration devices, can disallow operation with samplewell caps, or can be very expensive.

SUMMARY

According to various embodiments, an optical instrument that includes amulti-notch filter for quantitative monitoring nucleic acid sequenceamplification in an amplification apparatus. The instrument can possessimproved dynamic range, automatic selection of exposure time to extenddynamic range, automatic adjustment for drift, simplified operation,relatively low cost, and easy changing of optics to accommodatedifferent fluorescent dyes. According to various embodiments, theinstrument can be used in monitoring a PCR, isothermal amplification, orother nucleic acid sequence amplification or replication techniques.

According to various embodiments, a instrument is provided that includesan excitation light source, at least one reaction region capable ofretaining at least one respective sample that is capable of emittingemission beams along an emission beam path, a multi-notch filterdisposed along an excitation beam path between the excitation lightsource and the at least one reaction region, and a detector arrangedalong the emission beam path. The at least one reaction region caninclude a plurality of reaction regions, for example, 96 reaction wells.The instrument can further include a second multi-notch filter disposedalong an emission beam path between the at least one reaction region andthe detector.

According to various embodiments, and by way of example, an opticalinstrument including a multi-notch filter is provided for monitoringpolymerase chain reaction replication of DNA. The amplification can bein a reaction region or apparatus that includes a well, for example, athermal cycler block for holding at least one vial containing asuspension of ingredients for the reaction. The ingredients can includea fluorescent dye that fluoresces proportionately in presence of DNA.The instrument is capable of monitoring other nucleic acid sequenceamplification reactions, including isothermal amplification reactions.

According to various embodiments, the instrument can include anexcitation light source, device for directing light beams, a multi-notchfilter, a light detector, and device for processing data signals. Thelight source can emit a source beam including at least a primaryexcitation frequency that can cause the dye to fluoresce at an emissionfrequency. A first device can be disposed to be receptive of the sourcebeam to effect an excitation beam including the excitation frequency. Aprimary focusing device can be disposed to focus the excitation beaminto each suspension such that the primary dye can emit an emission beamincluding an emission frequency and an intensity representative of aconcentration of a target nucleic acid sequence in each suspension. Thefocusing device can be receptive of and can pass the emission beam. Asecond device can be disposed to be receptive of the emission beam fromthe focusing device so as to further pass the emission beam at theemission frequency to another focusing device that can focus theemission beam onto a detector. The detector can generate primary datasignals representative of the emission beam and thereby a correspondingconcentration of the target nucleic acid sequence in each vial. Aprocessor can be receptive of the primary data signals for computing anddisplaying the concentration of the target nucleic sequence.

According to various embodiments, the first device and the second devicetogether can include a beam splitter that can be receptive of the sourcebeam to effect the excitation beam, and receptive of the emission beamto pass the emission beam to the detector. The beam splitter can be amulti-notch beam splitter. The block can be configured to hold aplurality of vials. The focusing device can include a correspondingplurality of vial lenses each disposed over a vial. The focusing devicecan be disposed such that the emission beam can include individual beamsassociated with each vial. The focusing device can include a field lens,for example, a Fresnel lens. The field lens can be disposedcooperatively with the vial lenses to effect focusing of the excitationbeam into each suspension. The field lens can pass the individual beamsto the second device, for example, a beam splitter. According to variousembodiments, the detector can include an array of photoreceptorsreceptive of each individual beam to generate corresponding datasignals. The processing device can compute concentration of nucleic acidsequence in each vial.

According to various embodiments, the instrument can include amulti-notch excitation filter between the light source and the beamsplitter. The instrument can include a multi-notch emission filterbetween the beam splitter and the detector. The splitter and filters canbe associated with a selected primary dye in the suspension. In anotherembodiment, a filter module can contain the splitter and filters. Themodule can be removable from the housing for replacement with anothermodule associated with another selected primary dye. The excitationfilter can be a multi-notch filter. The emission filter can be amulti-notch filter. The beam splitter can be a multi-notch beamsplitter.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present teachings are exemplified in theaccompanying drawings. The teachings are not limited to the embodimentsdepicted, and include equivalent structures and methods as set forth inthe following description and known to those of ordinary skill in theart. In the drawings:

FIG. 1 is a schematic of an optical train for an optical instrumentaccording to various embodiments, associated with a nucleic acidsequence amplification reaction apparatus;

FIG. 1a illustrates an exemplary embodiment of a light source layout,for example, an organic light emitting diode (OLED) layout;

FIG. 1b illustrates an exemplary embodiment of a light source layout,for example, an OLED layout with varying color OLEDs stacked upon eachother;

FIG. 2 is a perspective of the instrument of FIG. 1 with a side panelremoved;

FIG. 3 is an exploded perspective of a module shown in FIG. 2;

FIG. 4 is a perspective of a reference member in the optical train ofFIG. 1;

FIG. 5 is a flow chart for computing DNA concentration from dataobtained with the instrument of FIG. 1;

FIG. 6 is a flow chart for determining exposure time for dataacquisition in operation of the instrument of FIG. 1 and forcomputations in the flow chart of FIG. 5;

FIG. 7 is a graph of extension phase data of fluorescence vs. cyclesfrom operation of the instrument of FIG. 1 with a PCR apparatus;

FIG. 8 is a flow chart for computing secondary data for computations inthe flow chart of FIG. 5; and

FIG. 9 is a flow chart for computing ratios between the plurality ofreference emitter segments of the reference member of FIG. 4.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended, to provide a further explanation of the variousembodiments of the present teachings.

DESCRIPTION

According to various embodiments, a instrument is provided that includesan excitation light source, at least one reaction region capable ofretaining at least one respective sample that is capable of emittingemission beams along an emission beam path, a multi-notch filterdisposed along an excitation beam path between the excitation lightsource and the at least one reaction region, and a detector arrangedalong the emission beam path. The at least one reaction region caninclude a plurality of reaction regions, for example, 96 reaction wells.The instrument can further include a second multi-notch filter disposedalong an emission beam path between the at least one reaction region andthe detector.

According to various embodiments, an instrument is provided thatincludes an excitation light source, at least one reaction well capableof retaining at least one respective sample that is capable of emittingemission beams along an emission beam path, a detector disposed alongthe emission beam path and capable of detecting emission beams emittedfrom the at least one reaction well, and a multi-notch filter spacedalong the emission beam path between the at least one reaction well andthe detector.

According to various embodiments, detectable emission beams can begenerated by a number of compounds, including many dyes. According tovarious embodiments, an optical instrument can be provided that includesa light source arranged to emit an excitation wavelength or wavelengthrange toward a region capable of retaining a sample, such that afluorescent dye, if present in the region, can be caused to fluoresce.The light source can provide excitation wavelength ranges thatcorrespond to respective excitation wavelength ranges of a plurality offluorescent dyes. A detector capable of detecting an emission wavelengthemitted from a fluorescing dye can be used to determine the absence orpresence of a component associated with the dye. For example, the dyescan include intercalating dyes, reporter dyes, free-floating dyes, andthe like.

According to various embodiments, PCR dyes can be used that onlyfluoresce when bound to a target molecule. Nucleic acid sequenceamplification dyes can also be attached to probes that also areconnected to quenchers, and the action of nucleic acid sequenceamplification enzymes will disassemble the dye-probe-quencher moleculecausing the dye to increase its fluorescence. According to variousembodiments, nucleic acid sequence amplification can be performed usinga variety of methods, for example, polymerase chain reaction (PCR),isothermal amplification reaction, well known in the art. When a PCRprocedure is used, for example, the number of unquenched dye moleculesdoubles with every thermal cycle. Fluorescing dyes are well known in theart, and any of a plurality of fluorescent dyes having variousexcitation wavelengths can be used. Examples of such dyes include, butare not limited to, Rhodamine, Fluoroscein, dye derivatives ofRhodamine, dye derivatives of Fluoroscein, 5-FAM™, 6-carboxyfluorescein(6-FAM™), VIC™, hexachloro-fluorescein (HEX™), tetrachloro-fluorescein(TET™), ROX™, and TAMRA™. Dyes or other identifiers that can be usedinclude, but are not limited to, fluorophores and phosphorescent dyes.Dyes can be used in combinations of two, three, four, or more dyes persample. According to various embodiments, the family of 5-FAM™, 6-FAM™,VIC™, TET™, and/or ROX™ dyes can be used to indicate the presence ofsample components.

According to various embodiments, various detectable markers can beused, in addition or in alternate, to dyes. Markers can include, forexample, fluorescing dyes, free-floating dyes, reporter dyes, probedyes, intercalating dyes, and molecular beacons. Dyes that fluorescewhen integrated into DNA can be intercalating dyes. Other dyes known as“reporter” dyes can attached to the ends of “probes” that have“quenchers” on the other end. A nucleic acid sequence amplificationreaction, for example, PCR, can result in the disassembly of theDye-Probe-Quencher molecule, so the reporter dye can emit an increasedamount of fluorescence. Reporter dyes are not attached in any way to thesample. Free floating dyes can be floating freely in solution. Otherfluorescing markers well know in the art can be utilized. According tovarious embodiments, molecular beacons can be single-stranded moleculeswith hairpins that preferentially hybridize with an amplified target tounfold. According to various embodiments, quantum dots can be used asmarkers also.

According to various embodiments, a method is provided that includes thedetection of a reference. For a reference, a fluorescent referencecompound can be caused to emit reference light in response to beingexposed to excitation beams. The reference can be disposed to bereceptive of a portion of the excitation beams from the excitationsource. A portion of the reference light can be passed by a seconddevice as a reference beam to the detector. The reference light can beused to generate reference signals for utilization in the computing ofthe concentration of DNA. In another embodiment, the reference membercan include a plurality of reference emitters. Each reference emittercan emit reference beams of different intensity in response to theexcitation beams. The processing device, for example, can then select areference set including the highest data signals that can be less than apredetermined maximum that can be less than the saturation limit. Amulti-notch filter can be used with reference emitters to filter thereference beams.

According to various embodiments, the detector can be operativelyconnected to the processing device. The detector can integrate emissionbeam input over a pre-selected exposure time for generating each set ofdata signals. The processing device, the detector, or a combinationthereof can include a saturation limit for the data signals. In anotherembodiment, the processing device can include an adjustment device forautomatically effecting adjustments in exposure time to maintain theprimary data within a predetermined operating range for maintainingcorresponding data signals less than the saturation limit. Theprocessing device, for example, can include a device for correcting theprimary data in proportion to the adjustments in exposure time.

According to various embodiments, the processor can computephotoreceptor data from the data signals for each photoreceptor. Theadjustment device can ascertain highest photoreceptor data. Theadjustment device can determine whether the highest photoreceptor datacan be less than, within or higher than the predetermined operatingrange. Based on such determination, the exposure time can be increased,retained or reduced so as to effect a subsequent exposure time formaintaining subsequent photoreceptor data within the predeterminedoperating range. The processing device can detect and/or discernexcitation, reference, and/or emission beams, any of which can be firstpassed through a multi-notch filter.

According to various embodiments, the terms “polynucleotide,” “nucleicacids,” “nucleotide,” “DNA,” and “RNA,” as used herein, are usedinterchangeably and can include nucleic acid analogs that can be used inaddition to or instead of nucleic acids. Examples of nucleic acidanalogs includes the family of peptide nucleic acids (PNA), wherein thesugar/phosphate backbone of DNA or RNA has been replaced with acyclic,achiral, and neutral polyamide linkages. For example, a probe or primercan have a PNA polymer instead of a DNA polymer. The 2-aminoethylglycinepolyamide linkage with nucleobases attached to the linkage through anamide bond has been well-studied as an embodiment of PNA and shown topossess exceptional hybridization specificity and affinity. An exampleof a PNA is as shown below in a partial structure with acarboxyl-terminal amide:

“Nucleobase” as used herein means any nitrogen-containing heterocyclicmoiety capable of forming Watson-Crick hydrogen bonds in pairing with acomplementary nucleobase or nucleobase analog, e.g. a purine, a7-deazapurine, or a pyrimidine. Typical nucleobases are the naturallyoccurring nucleobases such as, for example, adenine, guanine, cytosine,uracil, thymine, and analogs of the naturally occurring nucleobases,e.g. 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-azapurine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, pyrazolo[3,4-D]pyrimidines, “PPG”,and ethenoadenine.

“Nucleoside” as used herein refers to a compound consisting of anucleobase linked to the C-1′ carbon of a sugar, such as, for example,ribose, arabinose, xylose, and pyranose, in the natural β or the αanomeric configuration. The sugar can be substituted or unsubstituted.Substituted ribose sugars can include, but are not limited to, thoseriboses having one or more of the carbon atoms, for example, the2′-carbon atom, substituted with one or more of the same or differentCl, F, —R, —OR, —NR₂ or halogen groups, where each R is independently H,C₁-C₆ alkyl or C₅-C₁₄ aryl. Ribose examples can include ribose,2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose, 2′-fluororibose,2′-chlororibose, and 2′-alkylribose, e.g. 2′-O-methyl, 4′-α-anomericnucleotides, 1′-α-anomeric nucleotides, 2′-4′- and 3′-4′-linked andother “locked” or “LNA”, bicyclic sugar modifications. Exemplary LNAsugar analogs within a polynucleotide can include the followingstructures:

where B is any nucleobase.

Sugars can have modifications at the 2′- or 3′-position such as methoxy,ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy,phenoxy, azido, amino, alkylamino, fluoro, chloro and bromo. Nucleosidesand nucleotides can have the natural D configurational isomer (D-form)or the L configurational isomer (L-form). When the nucleobase is apurine, e.g. adenine or guanine, the ribose sugar is attached to theN⁹-position of the nucleobase. When the nucleobase is a pyrimidine, e.g.cytosine, uracil, or thymine, the pentose sugar is attached to theN¹-position of the nucleobase.

“Nucleotide” as used herein refers to a phosphate ester of a nucleosideand can be in the form of a monomer unit or within a nucleic acid.“Nucleotide 5′-triphosphate” as used herein refers to a nucleotide witha triphosphate ester group at the 5′ position, and can be denoted as“NTP”, or “dNTP” and “ddNTP” to particularly point out the structuralfeatures of the ribose sugar. The triphosphate ester group can includesulfur substitutions for the various oxygens, e.g. α-thio-nucleotide5′-triphosphates.

As used herein, the terms “polynucleotide” and “oligonucleotide” meansingle-stranded and double-stranded polymers of, for example, nucleotidemonomers, including 2′-deoxyribonucleotides (DNA) and ribonucleotides(RNA) linked by internucleotide phosphodiester bond linkages, e.g. 3′-5′and 2′-5′, inverted linkages, e.g. 3′-3′ and 5′-5′, branched structures,or internucleotide analogs. Polynucleotides can have associated counterions, such as H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺, Na⁺ and the like. Apolynucleotide can be composed entirely of deoxyribonucleotides,entirely of ribonucleotides, or chimeric mixtures thereof.Polynucleotides can be comprised of internucleotide, nucleobase andsugar analogs. For example, a polynucleotide or oligonucleotide can be aPNA polymer. Polynucleotides can range in size from a few monomericunits, e.g. 5-40 when they are more commonly frequently referred to inthe art as oligonucleotides, to several thousands of monomericnucleotide units. Unless otherwise denoted, whenever a polynucleotidesequence is represented, it will be understood that the nucleotides arein 5′ to 3′ order from left to right and that “A” denotesdeoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine,and “T” denotes thymidine, unless otherwise noted.

“Internucleotide analog” as used herein means a phosphate ester analogor a non-phosphate analog of a polynucleotide. Phosphate ester analogscan include: (i) C₁-C₄ alkylphosphonate, e.g. methylphosphonate; (ii)phosphoramidate; (iii) C₁-C₆ alkyl-phosphotriester; (iv)phosphorothioate; and (v) phosphorodithioate. Non-phosphate analogs caninclude compounds wherein the sugar/phosphate moieties are replaced byan amide linkage, such as a 2-aminoethylglycine unit, commonly referredto as PNA.

According to various embodiments, an optical instrument A can beutilized with or incorporated into a reaction apparatus B thatreplicates (“amplifies”) selected portions of DNA by nucleotideamplification reaction. The nucleotide amplification or replicationreaction can be a thermal or isothermal reaction. The nucleotideamplification reaction can be a polymerase chain reaction (“PCR”).

According to various embodiments, the reaction can occur in a reactionregion, for example, a well. Herein, “well” can mean a recess in asubstrate, a container, or a vial, for example. “Well” does not refer tocapillary electrophoresis channels in substrates or electrophoresiscapillaries.

The reaction apparatus can be conventional and can function withoutinterference from the instrument which can monitor the amount of nucleicacid in real time during replication. Suitable reaction apparatus aredescribed in U.S. Pat. Nos. 5,475,610 and 5,656,493.

In a thermal nucleotide amplification reaction, for example, PCR, thereaction apparatus (FIG. 1) can be conventional and can include two maincomponents, namely a thermal cycler block 1 with wells 1 a for holdingat least one vial 1 b containing a suspension of ingredients for thereaction, and a thermal cycle controller 1 c for cycling the temperatureof the block through a specified temperature program. The startingingredients of the aqueous suspension of sample materials can include a“seed” sample of a nucleic acid sequence, selected nucleic acid sequenceprimer strands, nucleic acid sequence elements, nucleic acid sequencefragments, enzymes, other chemicals, or a combination thereof. Theblock, for example, aluminum, can be heated and cooled in a prescribedcycle by an electrical device, liquid or air coolant devices, or acombination of these, or other devices to achieve the cycling. Thesuspensions in the vials can be cycled between two temperature phases soas to eftect the thermal nucleotide amplification reaction. These phasescan be a lower temperature extension phase for the PCR reaction of about60° C., which can be the phase where all of the nucleic acid sequencestrands have recombined into double strands. A high temperaturedenaturing phase at about 95° C. can be part of the PCR reaction, duringwhich the nucleic acid sequence can be denatured or split into singlestrands.

According to various embodiments, a sample can contain a fluorescent dyethat fluoresces proportionately and more strongly in the presence ofdouble stranded nucleic acid sequences to which the dye binds, forexample SYBR Green dye. The SYBR Green dye is available from MolecularProbes, Inc., Eugene, Oreg. The SYBR Green dye can fluoresce in thepresence of double stranded nucleic acid sequences. Another type offluorescent dye labeled “probes”, which can be DNA-like structures withcomplimentary sequences to selected nucleic acid sequence strandportions, can be used. Other dyes that have similar characteristics canbe utilized. As used herein and in the claims, the term “marker dye”refers to the type that binds to double stranded nucleic acid sequences,or to the probe type, or to any other type of dye that attaches tonucleic acid sequences so as to fluoresce in proportion to the quantityof a nucleic acid sequences. Samples can also contain an additional,passive dye (independent of the nucleic acid sequence) to serve as areference as described below. Under incidence of light including acorrect excitation frequency, a dye can fluoresce to emit light at anemission frequency. The emission frequency can be lower than that of theexcitation light.

According to various embodiments, the vials can be formed conically. Thevials can be formed in a plastic unitary tray. The tray can contain aplurality of vials, for example, 96 vials in an array of 12 by 8. Thetray can be removable from the block for preparations. A plastic unitarycover with caps 1 d for the vials can rest or attach over the vials toprevent contamination and evaporation loss. Other devices can be usedfor this function, for example, oil on the sample surface. If otherdevices are used to cover the tray, the vials caps may not be necessary.The caps can be transparent to light utilized in the instrument, forexample, the excitation frequency. The caps can be convex. The caps canface towards the light source or upwardly.

According to various embodiments, the monitoring instrument can bemounted over the block containing the vials. The instrument can beremovable or swing away for access to the vials. In the bottom of theinstrument, a platen 2 can rest over the vial caps or, if none, directlyover the vials. The platen can be metal, for example, aluminum. Theplaten can include an array of holes 2 a therethrough aligned with thevials. Each hole can have a diameter about the same as the vial topdiameter. The platen can have its temperature maintained by a filmheater or other devices for heating the platen. When caps are used, theheating can be controlled to prevent condensation under the caps withoutinterfering with nucleic acid sequence replication in the vials, forexample, by holding the platen at a slightly higher temperature than thehighest sample temperature that the thermal cycler reaches.

As depicted in FIG. 1, above each of the vials, a lens 2 b can bepositioned such that its focal point is approximately centered above thesuspension in the vial. Above these lenses, a field lens 3 can beprovided as a telecentric optical system. According to variousembodiments, the field lens 3 can be an aspherically corrected Fresnellens for minimal distortion. A neutral density pattern (not shown), tocorrect nonuniformities in illumination and imaging, can be mounted onor in proximity to the field lens, for example, to attenuate light inthe center of the image field. A folding optical mirror can beoptionally mounted, for example, at 45°, for convenient packaging. Thefolding optical mirror can be omitted, or other folding optics wellknown in the art, can be used. According to various embodiments, thefield lens and/or the vial lenses, can each include two or more lensesthat effect the required focusing. The word “lens” herein can includesuch multiplicities of lenses.

According to various embodiments, a light source 11 for a source beam 20of light can be provided. The light source can provide a flood light,for example, a 100 watt halogen lamp. The light source can provide lightat selective wavelengths, incoherent or incoherent. According to variousembodiments, the light source can be mounted at a focal distance of anellipsoid reflector 11 a which can produce a relatively uniform patternover the desired area. According to various embodiments, the reflectorcan be dichroic. The dichroic reflector can, for example, substantiallyreflect visible light and transmit infrared light, to restrict infraredfrom the other optical components, and from overheating the instrument.The cooling of the instrument can be aided by a heat reflecting mirror13 in the optical path. A mechanical or electronic shutter 12 can beused for blocking the source beam of the light source for obtaining darkdata. The type of light source can be a projection lamp, or a laser,with appropriate optical elements.

According to various embodiments, a beam splitter 6 can be disposed toreceive the source beam 20. In the present embodiment this can be adichroic reflector positioned, for example, at 45°, to reflect lightincluding an excitation frequency that can cause the marker dye tofluoresce at an emission frequency. The dichroic reflector can passlight including the emission frequency. Such a conventional opticaldevice can utilize optical interference layers to provide the specificfrequency response.

According to various embodiments, the beam splitter can be positioned toreflect the source beam to the folding mirror. The source beam can bereflected from the splitter as an excitation beam 22 includingsubstantially the excitation frequency. The excitation beam can befocused by the field lens 3 and then as separated beams 24 by the vial(well) lenses 2 b into the center of the vials. The marker dye can bethereby caused to emit light at the emission frequency. This light canbe passed upwardly, as depicted in FIG. 1, as an emission beam in theform of individual beams 26. The individual beams 26 can be reflectedfrom the folding mirror 5 to the beam splitter 6, which can pass theemission beam through to a detector 10.

According to various embodiments, the vial lenses 2 b and the field lens3 can together constitute a primary focusing device for focusing boththe excitation beam and the emission beam. According to variousembodiments, the field lens can be omitted from the focusing device.According to various embodiments, the vial lenses can be omitted fromthe focusing device. In another embodiment, an objective lens in thefield lens position can be used to focus the individual emission beamson the detector.

According to various embodiments, the beam splitter 6 can pass thesource beam as an excitation beam and reflect the emission beam, withappropriate rearrangement of the lamp and the detector. Angles otherthan 45° can be used depending on the beam splitter utilized. The beamsplitter can be used to form a more perpendicular reflection and passthrough. The beam splitter can split the optical paths for theexcitation beam and the emission beam. Other variations known in the artthat achieve this can also be suitable. The dichroic device can helpminimize the source light reaching the detector. A can also be used. Thenon-dichroic beam splitter can be less efficient as significant amountsof source light are reflected or transmitted in the wrong direction, forexample, to the detector, and/or lost.

According to various embodiments, to further filter the source light, anexcitation filter 7 can be disposed between the light source 11 and thebeam splitter 6. This can pass light including the excitation frequencywhile substantially blocking light including the emission frequency.Similarly, an emission filter 8 can be disposed between the beamsplitter and the detector, in this case between the splitter and adetector lens 9 in front of the detector. This filter can pass lightincluding the emission frequency while substantially blocking lightincluding the excitation frequency.

According to various embodiments, a lens can be used as a detector lensto focus the emission frequency. A focusing reflector can be used forthe detector lens. Such an emission focusing device, detector lens, orreflector, can be located after, as shown, or before the beam splitter.The emission focusing device can be on either side of the emissionfilter. The emission focusing device can be integrated into the primaryfocusing device. The field lens can be an objective lens, for example,that focuses the emission beam onto the detector.

According to various embodiments, suitable filters can be conventionaloptical bandpass filters utilizing optical interference films, eachhaving a bandpass at a frequency optimum either for excitation of thefluorescent dye or its emission. Each filter can have very highattenuation for the other (non-bandpass) frequency, in order to prevent“ghost” images from reflected and stray light. For SYBR Green dye, forexample, the excitation filter bandpass wavelength can center around 485nm, and the emission filter bandpass wavelength can center around 555nm. The beam splitter can transition from reflection to transmissionbetween these two, e.g. about 510 nm, so that light less than thiswavelength can be reflected and higher wavelength light can be passedthrough.

According to various embodiments, a light source can be used to provideexcitation beams to irradiate a sample solution containing one or moredyes. For example, two or more excitation beams having the same ordifferent wavelength emissions can be used such that each excitationbeam excites a different respective dye in the sample. The excitationbeam can be aimed from the light source directly at the sample, througha wall of a sample container containing the sample, or can be conveyedby various optical systems to the sample. An optical system can includeone or more of, for example, a mirror, a beam splitter, a fiber optic, alight guide, or combinations thereof.

According to various embodiments, one or more filters, for example, abandpass filter, can be used with a light source to control thewavelength of an excitation beam. One or more filters can be used tocontrol the wavelength of an emission beam emitted from an excited orother luminescent marker. One or more excitation filters can beassociated with a light source to form the excitation beam. One or morefilters can be located between the one or more light sources and asample. One or more emission filters can be associated with an emissionbeam from an excited dye. One or more filters can be located between thesample and one or more emission beam detectors.

According to various embodiments, a filter can be a single bandpassfilter or a multiple bandpass filter. As used herein, a bandpass filterand a passband filter can be used interchangeably. A multiple passbandfilter can be, for example, a multi-notch filter, a multi-Rugate filter,or simply a Rugate filter. A multiple passband filter can be used withan incoherent light source, for example, a halogen lamp, a white lightsource, and/or one or more Light Emitting Diode (LED), or Organic LEDsemitting light at different wavelengths. A multiple passband filter canbe used with a multiple laser-based light source emitting light atdifferent wavelengths. Examples of manufacturing Rugate filters andRugate beam splitters can be found in, for example, U.S. Pat. No.6,256,148 to Gasworth, which is incorporated herein by reference in itsentirety.

According to various embodiments, a multiple passband filter can be usedwith a dichroic beam splitter, a 50/50 beam splitter, a dichroic beamsplitter that has several “passbands,” or no beam splitter. A multiplebeam splitter can be coated at an angle, causing a variance in athickness across a filter substrate, to compensate for wavelength shiftwith an angle. A multiple passband filter can be formed by coatingdifferent light interference materials over respective areas of asubstrate used in a multiple passband filter manufacture.

According to various embodiments, a Rugate filter can be an example ofan interference coating based on the refractive index that variescontinuously in a direction, for example, perpendicular or 45 degrees tothe film plane. When the refractive index varies periodically within twoextreme values, a minus filter with high transmittance on either side ofthe rejection band can be made. Periodic Rugate filters can bemanufactured. In principle, a Rugate filter can be an interferencecoating based on a refractive index that varies continuously (not indiscrete steps) in the direction perpendicular to the film plane. Whenthe refractive index varies periodically and within two extreme values,it can be possible to design a rejection filter with high reflectance,approaching 99%, in the middle, and high transmission, approaching 99%,on either side of the rejection band. The Rugate filter can be designedto eliminate side-lobes, higher harmonics, and other significantparasitic losses. A Rugate filter overlay can have conjugatereflectance/transmittance characteristics matching the desired spectralresponse.

According to various embodiments, Rugate notch filters can userefractory metal oxides to achieve coatings with exceptional thermal andenvironmental stability. These filters can be used in place of othertypes of notch filters, particularly where durability and reliabilityare desired. Rugate notch filters are available from Barr Associates(Westford, Mass.). The Rugate notch filter can be used as edge filtersand beam splitters. Filter sizes or shapes are generally not limitationsfor the Rugate notch filter. The Rugate notch filter can provideenvironmental and thermal stability, a broad operating temperaturerange, narrow rejection bands, variety of shapes & sizes, highthroughput, low ripple, and/or a broad spectral range. More informationis available from, for example, www.barr-associates-uk.com,www.barrassociates.com/opticalfilters.php.

According to various embodiments, multi-notch filters can be made, forexample, with a measured blocking of O.D. 6 or better. Notch filterswith this type of deep blocking level at the light wavelength can alsoafford high transmission close to the light line.

According to various embodiments, excitation levels can increase whenmultiple dyes spaced apart spectrally are irradiated with excitationbeams. This can lead to less spectral crosstalk. The dye matrix,condition number, and/or deconvolution in a system can be improved. Theincreased excitation levels can provide higher signal levels. Highersignal levels can be seen during the utilization of dyes that emit inthe “red” spectrum. The dynamic range of the system can be improved. Thesystem can reduce the compensation for variation in the emission beamintensity for various dyes.

More broadly, the excitation filter and the beam splitter can togetherconstitute a first device disposed to be receptive of the source beam toeffect an excitation beam including the excitation frequency, and theemission filter and the beam splitter together constitute a seconddevice disposed to be receptive of the emission beam from the focusingdevice so as to pass the emission beam at the emission frequency to thedetector. Also, as mentioned above, the beam splitter alternatively canpass the source beam as an excitation beam and reflect the emission beamto the detector. In another aspect, the filters can be omitted, and thefirst device can be represented by the beam splitter effecting theexcitation beam from the source beam, and the second device can berepresented by the beam splitter passing the emission beam to thedetector.

According to various embodiments, the beam splitter can be omitted, andthe first device can constitute an excitation frequency, the seconddevice can constitute an emission filter for the emission frequency,where the light source and the detector can be side by side so that theexcitation and emission beams can be on slightly different optical pathsangularly. The source and detector need not actually be side by sidewith one or more folding mirrors. Thus any such arrangement forachieving the effects described herein can be deemed equivalent.According to various embodiments, the beam splitter can be capable ofpassing the excitation and emission beams through the field lens,allowing the excitation and emission beams to have a common opticalpath, fully or partially.

According to various embodiments, the beam splitter 5 in FIG. 1 can bereplaced with a 50/50 beam splitter, or a dichroic that has severalpassbands. A multi-notch filter, or a Rugate filter can be used asdichroic that has several passbands.

According to various embodiments, the light source can be a LightEmitting Diode (LED). The LED can be, for example, an Organic LightEmitting Diode (OLED), a Thin Film Electroluminescent Device (TFELD), ora Quantum dot based inorganic “organic LED.” The LED can include aphosphorescent OLED (PHOLED). As used herein, the terms “excitationsource” and “light source” are used interchangeably.

According to various embodiments, excitation beams emitted from thelight source can diverge from the light source at an angle ofdivergence. The angle of divergence can be, for example, from about 5°to about 75° or more. The angle of divergence can be substantially wide,for example, greater than 45°, yet can be efficiently focused by use ofa lens, such as a focusing lens.

According to various embodiments, a light source can contain one LightEmitting Diode (LED) or an array of LEDs. According to variousembodiments, each LED can be a high power LED that can emit greater thanor equal to about 1 mW of excitation energy. In various embodiments, ahigh power LED can emit at least about 5 mW of excitation energy. Invarious embodiments wherein the LED or array of LEDs can emit, forexample, at least about 50 mW of excitation energy, a cooling devicesuch as, but not limited to, a heat sink or fan can be used with theLED. An array of high-powered LEDs can be used that draws, for example,about 10 watts of energy or less, about 10 watts of energy or more. Thetotal power draw can depend on the power of each LED and the number ofLEDs in the array. The use of an LED array can result in a significantreduction in power requirement over other light sources, such as, forexample, a 75 watt halogen light source or a 150 watt halogen lightsource. Exemplary LED array sources are available, for example, fromStocker Yale under the trade name LED AREALIGHTS. According to variousembodiments, LED light sources can use about 1 microwatt of power orless, for example, about 1 mW, about 5 mW, about 25 mW, about 50 mW,about 1 W, about 5 W, about 50 W, or about 100 W or more, individuallyor when in used in an array.

According to various embodiments, a quantum dot can be used as a sourcefor luminescence and as a fluorescent marker. The quantum dot based LEDcan be tuned to emit light in a tighter emission bandpass, thus thequantum dot based LED can increase the efficiency of the fluorescentsystem. Quantum dots can be molecular-scale optical beacons. The quantumdot nanocrystals can behave like molecular LEDs (light emitting diodes)by “lighting up” biological binding events with a broad palette ofapplied colors. Quantum dots can provide many more colors thanconventional fluorophores. Quantum dots can possess many other verydesirable optical properties. Nanocrystal quantum dots can be covalentlylinked to biomolecules using standard conjugation chemistry. The quantumdot conjugate can then be used to detect a binding partner in a widerange of assays. According to various embodiments, streptavidin can beattached to quantum dots to detect biotinylated molecules in a varietyof assays. Quantum dots can also be attached to antibodies andoligonucleotides. Any assay that currently uses, for example,fluorescent-tagged molecules, colorimetric enzymes, or colloidal gold,can be improved with quantum dot nanocrystal-tagged conjugates. Anexemplary quantum dot implementation is available from Quantum DotCorporation of Haywood, Calif. under the trademark QDOT. Moreinformation about quantum dots and their applications can be found at,for example, www.qdot.com. U.S. Pat. Nos. 6,207,229, 6,251,303,6,306,310, 6,319,426, 6,322,901, 6,326,144, 6,426,513, and 6,444,143 toBawendi et al., U.S. Pat. Nos. 5,990,479, 6,207,392, and 6,423,551 toWeiss et al., U.S. Pat. No. 6,468,808 to Nie et al., and U.S. Pat. No.6,274,323 to Bruchez et al., describe a variety of biologicalapplications, methods of quantum dot manufacturing, and apparatuses forquantum dot nanocrystals and conjugates, all of which are incorporatedherein by reference in their entireties.

Quantum dots can provide a versatile probe that can be used in, forexample, in multiplex assays. Fluorescent techniques using quantum dotnanocrystals can be much faster than conventional enzymatic andchemiluminescent techniques, can reduce instrument tie-up, and canimprove assay throughput. Colorimetric or detected reflectancetechniques can be inferior to fluorescence and difficulties can ensuewhen multiplex assays are developed based on these materials. Quantumdots can absorb all wavelengths “bluer” (i.e., shorter) than theemission wavelength. This capability can simplify the instrumentationrequired for multiplexed assays, since all different label colors can beexcited with a single excitation source.

A Quantum dot based LED can emit light in an emission band that isnarrower than an emission band of a normal LED, for example, about 50%narrower or about 25% narrower. The Quantum dot based LED can also emitlight at an electrical energy conversion efficiency of about, 90% ormore, for example, approaching 100%. OLED films, including Quantum dotbased LEDs, can be applied to a thermal block, used for heating andcooling samples, in a fluorescence system without interfering with theoperation of the thermal block.

According to various embodiments, when an OLED is used, the OLED canhave any of a variety of sizes, shapes, wavelengths, or combinationsthereof. The OLED can provide luminescence over a large area, forexample, to luminescence multiple sample wells. Scatter or cross-talklight between multiple sample wells for this single OLED can be reducedby either overlaying a mask on the OLED or by patterning the luminescentin the OLED to operatively align with the multiple sample wells. TheOLED can be a low power consumption device. Examples of OLEDs in variousconfigurations and wavelengths are described in, for example, U.S. Pat.No. 6,331,438 B1, which is incorporated herein by reference in itsentirety. The OLED can include a small-molecule OLED and/or apolymer-based OLED also known as a Light-Emitting Polymer (LEP). Asmall-molecule OLED that is deposited on a substrate can be used. AnOLED that is deposited on a surface by vapor-deposition technique can beused. An OLED can be deposited on a surface by, for example,silk-screening. An LEP can be used that is deposited by, for example,solvent coating.

According to various embodiments, an OLED is used and can be formed fromone or more stable, organic materials. The OLED can include one or morecarbon-based thin films and the OLED can be capable of emitting light ofvarious colors when a voltage is applied across the one or morecarbon-based thin films.

According to various embodiments, the OLED can include a film that islocated between two electrodes. The electrodes can be, for example, atransparent anode, a metallic cathode, or combinations thereof. Severalseparate emission areas can be stimulated between a single set ofelectrodes where simultaneous illumination of the separate emissionareas is required. According to such embodiments, only one power andcontrol module could be required for several apparent light sources. TheOLED film can include one or more of a hole-injection layer, ahole-transport layer, an emissive layer, and an electron-transportlayer. The OLED can include a film that is about one micrometer inthickness, or less. When an appropriate voltage is applied to the film,the injected positive and negative charges can recombine in the emissivelayer to produce light by means of electroluminescence. The amount oflight emitted by the OLED can be related to the voltage applied throughthe electrodes to the thin film of the OLED. Various materials suitablefor fabrication of OLEDs are available, for example, from H.W. SandsCorp. of Jupiter, Fla. Various types of OLEDs are described, forexample, in U.S. Pat. No. 4,356,429 to Tang, U.S. Pat. No. 5,554,450 toShi et al., and U.S. Pat. No. 5,593,788 to Shi et al., all of which areincorporated herein by reference in their entireties.

According to various embodiments, an OLED can be used and produced on aflexible substrate, on an optically clear substrate, on a substrate ofan unusual shape, or on a combination thereof. Multiple OLEDs can becombined on a substrate, wherein the multiple OLEDs can emit light atdifferent wavelengths. Multiple OLEDs on a single substrate or multipleadjacent substrates can form an interlaced or a non-interlaced patternof light of various wavelengths. The pattern can correspond to, forexample, a sample reservoir arrangement. One or more OLEDs can form ashape surrounding, for example, a sample reservoir, a series of samplereservoirs, an array of a plurality of sample reservoirs, or a sampleflow path. The sample path can be, for example, a channel, a capillary,or a micro-capillary. One or more OLEDs can be formed to follow thesample flow path. One or more OLEDs can be formed in the shape of asubstrate or a portion of a substrate. For example, the OLED can becurved, circular, oval, rectangular, square, triangular, annular, or anyother geometrically regular shape. The OLED can be formed as anirregular geometric shape. The OLED can illuminate one or more samplereservoirs, for example, an OLED can illuminate one, two, three, four,or more sample reservoirs simultaneously, or in sequence. The OLED canbe designed, for example, to illuminate all the wells of a correspondingmulti-well array.

According to various embodiments, one or more excitation filters can beincorporated into the OLED substrate, thus eliminating additionalequipment and reducing the amount of space needed for an optical system.For example, one or more filters can be formed in a layer of a substrateincluding one or more OLEDs and a layer including a sample flow path.The wavelength emitted by the OLED can be tuned by printing afluorescent dye in the OLED substrate, as taught, for example, by Hebneret al. in “Local Tuning of Organic Light-Emitting Diode Color by DyeDroplet Application,” APPLIED PHYSICS LETTERS, Vol. 73, No. 13 (Sep. 28,1998), which is incorporated herein by reference in its entirety. Whenusing multiple emission lines in an OLED, the OLED can be used incombination with a multiple notch emission filter.

According to various embodiments, an OLED can be substituted in place ofany of the systems, devices, or apparatuses where an LED is shown. TheOLED light source can have several OLED films stacked and operativelydisposed such that several wavelengths of excitation beams can traversethe same optical path to illuminate the sample well. Several OLEDsforming excitation beams of the same wavelength can be stacked toprovide higher output to illuminate the sample well.

According to various embodiments, a sample well can be placed in betweenan excitation source and a detector. The sample well can be a microcard, for example, a microtiter card, such as a 96-well microtiter card.The excitation source can be, for example, an OLED, standard LED, orcombination thereof.

According to various embodiments, the light source can be a Solid StateLaser (SSL) or a micro-wire laser. The SSL can produce monochromatic,coherent, directional light and can provide a narrow wavelength ofexcitation energy. The SSL can use a lasing material that is distributedin a solid matrix, in contrast to other lasers that use a gas, dye, orsemiconductor for the lasing source material. Examples of solid statelasing materials and corresponding emission wavelengths can include, forexample: Ruby at about 694 nm; Nd:Yag at about 1064 nm; Nd:YVO4 at about1064 nm and/or about 1340 nm and which can be doubled to emit at about532 nm or about 670 nm; Alexandrite at from about 655 nm to about 815nm; and Ti:Sapphire at from about 840 nm to about 1100 nm. Micro-wirelasers are lasers where the wavelength of an excitation beam formed bythe laser can be tuned or adjusted by altering the size of a wire.According to various embodiments, other solid state lasers known tothose skilled in the art can also be used, for example, laser diodes.The appropriate lasing material can be selected based on the fluorescingdyes used, the excitation wavelength required, or both.

According to various embodiments, if a SSL is used, the laser can beselected to closely match the excitation wavelength of a fluorescentdye. The operating temperature of the system can be considered inselecting an appropriate SSL. The operating temperature can be regulatedor controlled to change the emitted wavelength of the SSL. The lightsource for the laser can be any source as known to those skilled in theart, such as, for example, a flash lamp. Useful information aboutvarious solid state lasers can be found at, for example,www.repairfaq.org/sam/lasersl.htm. Examples of solid state lasers usedin various systems for identification of biological materials can befound in, for example, U.S. Pat. No. 5,863,502 to Southgate et al. andU.S. Pat. No. 6,529,275 B2 to Amirkhanian et al.; both of which areincorporated herein by reference in their entireties.

According to various embodiments, various types of light sources can beused singularly or in combination with other light sources. One or moreOLEDs can be used with, for example, one or more non-organic LEDs, oneor more solid state lasers, one or more halogen light sources, orcombinations thereof.

FIG. 1a is a bottom view that illustrates an OLED layout 400 that can beused as a light source, together with a plurality of photodiodedetectors 412, according to various embodiments. The OLED layout 400 caninclude a plurality of OLED well lamps 402, each positioned, when inoperation, above a respective well of a multi-well sample well array.Each OLED material well lamp 402 can be connected to, or integrallyformed with, a respective connection arm 404 that leads to a layoutterminal 406. Each layout terminal can be connected to or integrallyformed with the respective connection arms 404 branching from the layoutterminal.

According to various embodiments, the connection arms 404 can branch offof side terminals 406 and 408. The OLED layout can be connected torespective opposite electrical connections, for example, oppositeterminals of a power supply. The OLED layout can be connected to thepower supply through leads arranged at opposite corners of the OLEDlayout. The power supply can include or be connected to one or more of aswitch, a meter, an oscillator, a potentiometer, a detector, a signalprocessing unit, or the like. Alternatively, or additionally, connectionarms 404 can each include a wire or electrical lead in the form of, forexample, a metal wire. The OLED layout can include a plurality ofindividually addressable OLED lighting elements (not shown) with aseparate lead connected to each lighting element. The wiring, leads,terminals, connection arms, and the like can be implemented in, forexample, a substrate or a film. An OLED layout control unit 410 can beused to supply power and control the OLED layout 400. A plurality ofdetectors 412 can be electrically connected to a detector control unit416 through respective detector leads 414 as shown.

According to various embodiments, the plurality of detectors can bearranged, for example, centered, on the plurality of OLED well lamps402, on the sides of well lamps that face respective sample wells,and/or when operatively positioned adjacent a multi-well sample wellarray. The detectors can be configured to detect light emitted from thesample wells of a sample well array, without being flooded or bleachedout by the respective OLED well lamps. For example, a mask material canbe disposed between the detectors and the respective OLED well lamps.The detector 412 can be formed in the same substrate as the OLED lamp.

According to various embodiments, the exemplary OLED layout shown inFIG. 1a is shaped to be aligned with a 24 well sample well array. Otherembodiments of OLED layouts using various shapes and various numbers ofwell lamps are within the scope of the present teachings.

According to various embodiments, each well lamp 402 can include, forexample, four individual lamps or OLED layers, capable of producingexcitation wavelengths at four different frequencies.

According to various embodiments, the OLED layout can be constructed ofa unitary or multi-part construction, of molded material, of stampedmaterial, of screen printed material, of cut material, or the like.

FIG. 1b illustrates an exemplary embodiment of a light source layout. AnOLED layout 450 can include varying color OLEDs 452, 454, and 456stacked upon each other. The layout can be useful for a compact lightsource design capable of forming excitation beams at varyingwavelengths. The OLEDs 452, 454, and 456 can be transparent, allowingexcitation beams from each OLED to pass through any other OLED so as tobe directed towards a sample. The OLEDs 452, 454, and 456 can emitdifferent colors, same colors, or a combination thereof depending on thecolor intensity and variety required. The OLEDs 452, 454, and 456 canshare an electrode, for example, a cathode. One electrode, for example,an anode, for powering each of the OLEDs 452, 454, and 456 can beconnected in electrical isolation from each respective anode to acontrol unit (not shown) if the capability to independently activateeach of the OLEDs 452, 454, and 456 is desired. The OLEDs 452, 454, and456 can electrically share one electrode, two electrodes, or noelectrodes. Any number of OLEDs can be stacked, for example, two OLEDs,three OLEDs, four OLEDs, or more OLEDs, to form a light source, arespective light source, or an array of light sources.

According to various embodiments, multiple excitation wavelengths can beused to detect multiple sample components. According to variousembodiments, the apparatus and method can be adapted for use by anysuitable fluorescence detection system. For example, various embodimentsof the apparatus and method can be used in a sequencing system withsingle or multiple samples, for example, in a nucleic acid sequenceamplification reaction, in a sequencing detection system.

According to various embodiments, the beam splitter 6, the excitationfilter 7 and the emission filter 8 can be affixed in a module 30 (FIG.2) that can be associated with a selected primary dye for thesuspension. The module can be removable from the housing 32 of theinstrument A for replacement with another module containing differentbeam splitter and filters associated with another selected primary dye.The instrument can include a lamp subhousing 33 and a camera subhousing35.

In an example (FIG. 3), and according to various embodiments, eachmodule can include a mounting block 34 with a flange 36 that can beaffixable to the housing with a single screw 38. The beam splitter 6 canbe held at 45° in the block with a frame 40 and screws 42. The emissionfilter 8 can mount, for example, with glue, into the block. Theexcitation filter 7 can be similarly mounted into a mounting member 44that can be held by screws 46 to the block. With the module in place,the instrument can be closed up with a side plate 47 that can be screwedon. Positioning pins (not shown) ensure repeatable alignment. Thereplacement module can include the same mounting block and associatedcomponents, with the beam splitter and filters replaced.

According to various embodiments, the detector lens 9 (FIG. 1) can becooperative with the vial lenses 2 b and the field lens 3 to focus theindividual beams on the detector 10. The lens can be large aperture, lowdistortion, and minimum vignetting.

According to various embodiments, the detector preferably can be anarray detector, for example, a charge injection device (CID) or,preferably, a charge coupled device (CCD). A conventional video cameracontaining a CCD detector, the detector lens and associated electronicsfor the detector can be suitable, such as an Electrim model 1000L whichincludes 751 active pixels horizontal and 242 (non-interlaced) activepixels vertical. This camera can include a circuit board that candirectly interface to a computer ISA bus. No frame grabber circuitry isrequired with such a camera. Essentially any other digital imagingdevice or subsystem can be used or adapted that can be capable of takingstill or freeze-frame images for post processing in a computer.

According to various embodiments, a detector with a multiplicity ofphotoreceptors (pixels) 78 can be preferable if there is a plurality ofvials. The detector can provide separate monitoring of each vial. Inanother embodiment, a scanning device can be used with a singlephotodetector, for example, by scanning the folding mirror and using asmall aperture to the detector. A simple device such as aphotomultiplier can be used, if there is only one vial. A CCD canreceive light for a selected integration period and, afteranalog/digital conversion, can read out digital signal data at a levelaccumulated in this period. The integration can be effectivelycontrolled by an electronic shutter. A frame transfer circuit can bedesirable. Signal data can be generated for each pixel receiving theindividual beams of emitted light from the vials.

According to various embodiments, the instrument can include afluorescent reference member 4 that can emit a reference light inresponse to the excitation beam. The reference member can be formed of aplurality of reference emitters, for example, 6, each emitting areference beam of different intensity in response to the excitationbeam. The range of these intensities can approximate the range ofintensities expected from the marker dye in the vials; for example, eachsegment can be separated in brightness by about a factor of 2.5. Thereference member can be disposed to receive a portion of the excitationbeam from the beam splitter. A good location for the reference membercan be adjacent to the field lens, so that the optical paths associatedwith the member approximate those of the vials. Most of the referencelight can pass back through the beam splitter as a reference beam to thedetector. The detector pixels can receive the emission beam to generatereference signals for utilization along with the data signals in thecomputing of the concentration of the nucleic acid sequence.

According to various embodiments, the reference member 4 (FIG. 4) caninclude a plastic fluorescent strip 4 a. The reference member 4 caninclude a neutral density filter 4 b mounted over the fluorescent strip4 a. The reference member 4 can include an air space 4 h the fluorescentstrip 4 a, such that a portion of the excitation beam and the referencebeam can be attenuated by the neutral density filter. The neutraldensity filter can have a series of densities 4 c to effect theplurality of reference emitters (segments), each emitting a referencebeam of different intensity. A heating strip 4 d and an aluminum strip 4g can be mounted in a trough 4 e on the bottom thereof, as depicted inFIG. 4, to smooth the heating. The fluorescent strip can be mounted onthe aluminum strip over the heating strip. To prevent heat loss, thisassembly can be covered by a transparent Plexiglas window (not shown, soas to display the varying density filter). To help maintain constantfluorescence, the heating strip can be controlled to maintain thefluorescent strip at a constant temperature against the thermal cyclesof the cycler block and other effects. This can be done because mostfluorescent materials change in fluorescence inversely with temperature.

According to various embodiments, the computer processor 14 (FIG. 1) canbe a conventional PC. The computer programming can be in conventionalcomputer language, for example, “C”. Adaptations of the programming forthe present teachings can be readily recognized and achieved by thoseskilled in the art. The processor can selectively processes signals frompixels receiving light from the vials and the reference emitters. Theprocessor can selectively ignore surrounding light signals. According tovarious embodiments, the programming can include masking to define thepixel regions of interest (ROI), for example, as disclosed in co-pendingprovisional patent application Ser. No. 60/092,785 filed Jul. 14, 1998of the present assignee. Mechanical alignment of the optics can benecessary to cooperatively focus the beams into the programmed regionsof interest. The analog data signals can be fed to the processor throughan analog/digital (A/D) device 15 which, for the present purpose, can beconsidered to be part of the processor. A saturation level can beproscribed by the detector, the A/D, the CCD dynamic range, or acombination thereof can be matched to the A/D dynamic range. A suitablerange can be, for example, 8 bits of precision (256 levels). The CCDamplifier offset can be set so that the dark signal output of the CCD(with the shutter 12 closed) can be within the A/D range. The processorcan instruct the detector with selected exposure time to maintain theoutput within the dynamic range.

In operation, fluorescence data can be taken from the plurality of vials(e.g. 96 regions of interest) and from the reference emitter segments,for each cycle in a nucleic acid sequence replication reaction. For PCR,the number of thermal cycles can be from about 40 cycles to about 50cycles. Two data sets can be taken (FIG. 5) for each cycle during theextension phase of the PCR reaction at about 60° C., which can be thephase where all of the nucleic acid sequence strands have recombinedinto double strands. One set can be normal primary data 50 (along withreference data described below) and the other set can be dark signaldata 51 with the mechanical shutter closed. Both digital data sets 50,51 can be converted by the A/D 15 from respective analog data signals48, 49 from the detector. The dark can be subtracted 55 from the normal,to yield dark-corrected data 57. In a simple procedure, the subtractioncan be pixel by pixel. According to various embodiments, total dark foreach region of interest can be subtracted from corresponding totalfluorescence data. In another embodiment, multiple exposures can becollected during each exposure period, for example, 4 or 8 exposures, inorder to increase the effective dynamic range of the instrument. Thiscan be done by collecting multiple normal exposures and dark signal datafor each pixel, subtracting each respective dark image from the normaldata, then adding the subtracted data together to yield the primarydata. This can improve the statistical validity of the image data andincreases its effective dynamic range.

Data can be taken simultaneously from the reference strip whichincludes, for example, 6 segments together with the 96 vials for a totalof 102 regions of interest. According to various embodiments, theprocessing device can provide for automatic adjustment of the exposuretime to maintain the data signals within a predetermined operating rangethat can be less than the saturation limit during the nucleic acidsequence replication sequence, for example, 35% to 70% of saturation.Computations for nucleic acid sequence concentration can includecorrections in proportion to adjustments in exposure time (FIG. 6).Signal data 50, 51 from each exposure 52, 53 can be obtained during apreviously determined exposure time 54 by totaling the pixel countswithin each region of interest (ROI).

To provide the time adjustments, the highest signal data 56 can besearched out 58 from the corresponding data signals 50. The highestsignal data 56 can be data from one or more of the highest pixelreadings, such as the three highest contiguous pixels. It can bedetermined from a comparison 62 whether the highest signal data can beless than, within, or higher than the selected operating range 60. Basedon such determination, the exposure time can be adjusted 64, i.e.increased, retained, or reduced, to obtain the subsequent exposure time66. A reference time 68 (FIG. 5) can be selected which can be, forexample, an initial time or a fixed standard time, for example, 1024 ms.The dark-corrected data 57 can be time-corrected 69 to yield correctedprimary data 71, that can be divided by ratio of actual exposure time tothe reference time. The first several cycles can be out of range, andthereafter a useful fluorescence curve can be obtained (FIG. 7).

According to various embodiments, for the reference emitter, the pixelsreceiving light from the reference strip 4 (FIGS. 1 and 4) referencedata signals 73 can be generated and can be converted by the A/D 15 toreference data 72. Selected reference data 74 from a specific referencesegment 4 c (FIG. 4) can be selected 76. Reference data 74 can have thehighest signal strength that can be less than a predetermined maximum 77that, in turn, can be less than the saturation limit, for example, 70%.A next dimmer segment can be also selected 75, and the selectedreference data 74 can include the data from that segment. The dark data51 can be subtracted 78 from the reference data 74, and the darkcorrected data 80 can be adjusted 84 for exposure time 54 to yieldadjusted reference data 82.

According to various embodiments, the data 82 can include dark correcteddata 82′ for the highest segment and dark corrected data 82″ for thenext dimmer segment (FIG. 9). The ratios of brightness between eachsegment can be computed 89 and can be built up over the course of datacollection. Each time data is collected, the ratio between the highestand next dimmer segment can be calculated. As different optimum segmentscan be selected on succeeding data collections, a table of ratios 85 canbe assembled. According to various embodiments, these ratios can becollected and calculated in advance. This adjusted reference data 82′(from data 82, FIG. 5) can be utilized for computing normalizedreference data 88 which can be normalized 86 in real time as a ratio toreference data 90 from an initial or other selected previous cycle inthe nucleic acid sequence replication sequence by working back with theratios 85. The normalized reference data can be utilized on thecorrected primary data 71 in a normalization computation 92 to providedrift normalized primary data 94 by dividing the primary data by thenormalized reference data. This can correct for instrument drift duringthe monitoring. Nucleic acid sequence concentration 96 can then becomputed 98 from a stored calibration factors 99, and can be determinedby running standard known nucleic acid sequence concentrations todetermine the slope and intercept of a line relating startingconcentration to the starting cycle of the growth curve (FIG. 7) astaught in the aforementioned article by Higuchi and U.S. Pat. No.5,766,889. Further normalization 118, 120 and baseline correction122-130 are discussed below.

Extension phase data for a typical replication sequence could look likeFIG. 7, plotted for each replication cycle. If desired, the data can becorrected for dye bleaching and/or other sample chemical effects bynormalizing to sample vials containing samples with the same dye andwith nucleic acid sequence amplification prevented chemically.

According to various embodiments, the sample can contain one or moretypes of dye molecules that serve as a “passive” reference includingsome fluorescence in the same wavelength range as the nucleic acidsequence binding dye. This reference dye can be a nucleic acid sequence,for example, a nucleic acid sequence labeled with Rhodamine andFluorescein dye derivatives. A suitable reference can be Rox dye fromPerkin-Elmer Applied Biosystems. These passive dye molecules do not takepart in the replication reaction, so that their fluorescence can besubstantially without influence from the nucleic acid sequence. Theirfluorescence can remain constant during the reaction. This fluorescencecan be used to normalize the fluorescence from the nucleic acid sequencebinding dye with a standard concentration of passive dye included in theingredients of at least one vial. The passive dye can be in every vial.

According to various embodiments, the source beam includes a secondaryexcitation frequency that can cause the passive dye to fluoresce at asecondary frequency. The passive dye can thereby emit a secondary beamdirected to the detector to generate corresponding secondary datasignals. The processor can be receptive of the secondary data signalsfor computing secondary data representative of standard concentration.These data can be used to normalize the primary data, so that theconcentration of the nucleic acid sequence can be normalized to thestandard concentration of passive dye after correcting computations ofconcentration of the nucleic acid sequence in proportion to adjustmentsin exposure time, and in conjunction with the normalization for drift.According to various embodiments, the secondary excitation frequency canbe identical to the primary excitation frequency. The passive dye canfluoresce such that the emitted secondary beam can be substantially atthe emission frequency. The primary data signals can be generated duringeach extension phase of cycling of the thermal cycler block when thenucleic acid sequence can be recombined and correspondingly primary dyeemission can be maximized. The secondary data signals can be generatedduring each denature phase of cycling of the thermal cycler block whenthe nucleic acid sequence can be denatured and correspondingly primarydye emission can be minimized. Thus data signals for the primary phasecan be substantially representative of the nucleic acid sequenceconcentration, and data signals for the secondary phase can besubstantially representative of the standard concentration of passivedye.

According to various embodiments, the dark and normal data can be takenfor the vial samples and the reference strip, and the dark can besubtracted from the normal fluorescence data. This dark and normal dataset can be taken during the extension phase of the replication reactionat about 60° C., which can be the phase where all of the nucleic acidsequence strands have recombined into double strands. During this phase,the fluorescence from the nucleic acid sequence binding dye can bemaximized, and the fluorescence from the passive reference molecules canbe superimposed but can be much smaller. A separate dark and normal dataset can be taken during the high temperature (about 95° C.) denaturingphase, during which the nucleic acid sequence can be denatured or splitinto single strands. During this phase, the fluorescence of the nucleicacid sequence binding dye can be minimized, and can be almostnon-existent, because the nucleic acid sequence is not double strandedand the fluorescence of the dyes used can have a large decrease influorescence with increased temperature. Therefore, the denaturing phaseimages substantially can contain reference fluorescence from the passivereference molecules. The dark-corrected reference (denaturing) data set,after correction for measured temperature dependence, can be subtractedfrom the dark-corrected nucleic acid sequence binding dye data set, orcan be deemed insignificant for the normal data set.

According to various embodiments, the passive reference dye labeledmolecules can be imaged by taking the additional images, for eachreplication or amplification cycle, using a separate optical band passfilter that rejects wavelengths emitted by the nucleic acid sequencebinding dye while accepting wavelengths from the passive reference dye.This data can be functionally equivalent to the denature data.

Illustrating operation for the denature phase (FIG. 8), respectivenormal and dark data signals 102, 104 can be obtained in the same manneras for the primary data, with normal exposure 52′ and closed shutter53′. Exposure time 106 can be the same as for an adjacent extensionphase in the sequence, or can be determined from a previous denaturephase run (as described with respect to FIG. 7), or can be apredetermined suitable time for all denature phases in the sequence. TheA/D 15 can convert the signals to secondary data 108 and dark data 110.The dark can be subtracted 55′ from the secondary to yielddark-corrected data 112 which can be further corrected 69′ with areference time 114 and the actual exposure time 106 that can yieldcorrected secondary data 116.

The extension cycle, drift normalized primary data 94 then can benormalized 118 by dividing by the average of a selected number ofcycles, for example, 10, for the denature phase corrected secondary data116 that can produce further normalized fluorescence data or furthernormalized data 120, which can remove sample well to well non-uniformityeffects. Cycle by cycle division can be used in place of an average.According to various embodiments, the secondary data can be applied tothe corrected primary data 71 before or after drift normalization.Baseline samples can be selected 122 and can be averaged 124 to producebaseline data 126. The further normalized data 120 can be then divided128 by the baseline data to yield baseline corrected data 130. Thesebaseline samples can be selected so as to be before the PCR growthexceeds the nearly horizontal base line portion of the curve in FIG. 7.Selected baseline cycles can be, for example, cycles 6 through 15. Afterfurther normalization 118, the further normalized data 118 can be usedto compute 98 a DNA concentration 96. The trend of these same baselinesamples, for example, least squares regression line, can be subtractedfrom the normalized extension cycle data, to produce data that caninclude a flat base line at zero. This data set can then be processedusing established or other desired amplification methods to calculatethe amount of starting copies of an nucleic acid sequence. A simpleprocedure can be to extrapolate for the inflection point at thetransition from flat to rising. A more sophisticated procedure isdescribed in the aforementioned U.S. Pat. No. 5,766,889.

According to various embodiments, the data can be used for variouspurposes, for example, quantitative monitoring of the reaction,determination of replicated nucleic acid sequence concentration, ordetermination of the starting amount. The instrument also be used simplyto display whether replication is taking place during a sequence, or hastaken place, for example, with or without normalizations and othercorrections.

Various embodiments of the teachings are described herein. The teachingsare not limited to the specific embodiments described, but encompassequivalent features and methods as known to one of ordinary skill in theart. Other embodiments will be apparent to those skilled in the art fromconsideration of the present specification and practice of the teachingsdisclosed herein. It is intended that the present specification andexamples be considered as exemplary only.

What is claimed is:
 1. An imaging optical instrument, comprising: anapparatus having a surface configured to hold a two-dimensional array ofspaced-apart reaction regions; an excitation light source comprising atleast two, individually addressable light elements, the excitation lightsource configured to provide at least two excitation beams havingdifferent excitation wavelength ranges; a beam splitter comprising adichroic beam splitter having two passbands, the beam splitterconfigured to direct the excitation beams to produce emission beamscomprising at least two different emission wavelength ranges; adetector; an imaging system comprising a lens configured to direct theexcitation beams to simultaneously illuminate the two-dimensional arrayof spaced-apart reaction regions and to direct the emission beams ontothe detector to produce two-dimensional images of the spaced-apartreaction regions; wherein the beam splitter is configured to reflectlight from each of the light elements and to transmit light from theemission beams through the beam splitter and to the detector.
 2. Theoptical instrument of claim 1, further comprising an excitation filterdisposed along an excitation path between the excitation light sourceand the beam splitter, the excitation filter comprising a multiplebandpass filter and configured to transmit light within the differentexcitation wavelength ranges.
 3. The instrument of claim 1, furthercomprising a Fresnel lens disposed along an excitation path from theexcitation light source to the surface and along an emission path fromthe surface to the detector.
 4. The instrument of claim 1, furthercomprising a mirror disposed along an excitation path from theexcitation light source to the surface.
 5. The instrument of claim 1,further comprising a plurality of spaced-apart reaction regions disposedon or near the surface, at least some of the reaction regions comprisingat least one respective sample including a fluorescent compoundconfigured to produce an emission beam in response to at least one ofthe excitation beams.
 6. The instrument of claim 5, wherein thefluorescent compound comprises a dye, marker, or label.
 7. Theinstrument of claim 5, further comprising a plurality of detectors todetect the emission beams from the reaction regions.
 8. The instrumentof claim 1, wherein the beam splitter comprises a Rugate filter.
 9. Theinstrument of claim 1, wherein the beam splitter is a dichroic beamsplitter.
 10. The instrument of claim 1, further comprising a processingdevice, operatively connected to the detector, the processing deviceconfigured to adjust exposure conditions of the detector to maintain adata signal from the detector within a predetermined operating range.11. The instrument of claim 1, wherein the excitation light sourcecomprises at least two lasers.
 12. The instrument of claim 1, whereinthe at least two light elements comprises at least two light emittingdiodes (“LEDs”).
 13. The optical instrument of claim 12, wherein the atleast two LEDs draw about 10 watts of energy or more.
 14. The instrumentof claim 1, wherein the detector comprises a charge coupled device (CCD)or a charge injection device (CID).
 15. The instrument of claim 1,wherein the detector comprises a plurality of photodiode detectors. 16.The instrument of claim 1, wherein the at least two light elementscomprises a plurality of light emitting diodes having differentwavelength ranges, the beam splitter being configured to reflect lightfrom each of the light emitting diodes and transmit light from theemission beams.
 17. The instrument of claim 1, wherein the detector is asingle array detector configured to receive emission beams from morethan one of the reaction regions.
 18. The optical instrument of claim 1,further comprising a thermal controller for controlling a temperature ofa block.
 19. An imaging optical instrument, comprising: an apparatushaving a surface configured to hold a two-dimensional array ofspaced-apart reaction regions; an excitation light source comprising atleast two, individually addressable light elements, the excitation lightsource configured to provide at least two excitation beams havingdifferent excitation wavelength ranges and to direct the excitationbeams to simultaneously illuminate more than one of the reaction regionsso as to produce a plurality of emission beams; a beam splittercomprising a dichroic beam splitter having at least two passbandscorresponding to the different excitation wavelength ranges; a detector;an imaging system comprising a lens configured to direct the excitationbeams to simultaneously illuminate the two-dimensional array ofspaced-apart reaction regions and to direct the emission beams onto thedetector to produce two-dimensional images of the spaced-apart reactionregions; wherein the beam splitter is (a) configured to transmit lightfrom each of the light elements through the beam splitter and to reflectlight within each of the at least two different emission wavelengthranges or (b) configured to transmit the at least two excitation beamshaving the different excitation wavelength ranges through the beamsplitter and to reflect light within each of the at least two differentemission wavelength ranges.
 20. An optical instrument, comprising: anapparatus having a region configured to hold a two-dimensional array ofspaced-apart reaction regions; an excitation light source comprising atleast two, independently activated light elements, the excitation lightsource configured to provide at least two excitation beams havingdifferent colors and to direct the excitation beams to simultaneouslyilluminate more than one of the reaction regions; a beam splittercomprising a dichroic beam splitter having two passbands, the beamsplitter configured to direct the excitation beams to the more than onereaction regions so as to provide emission beams comprising at least twodifferent colors; a detector; an imaging system comprising a lensconfigured to direct the excitation beams to simultaneously illuminatethe two-dimensional array of spaced-apart reaction regions and to directthe emission beams onto the detector to produce two-dimensional imagesof the spaced-apart reaction regions; wherein the beam splitter isconfigured to reflect light from each of the light elements and totransmit light from the emission beams through the beam splitter and tothe detector.
 21. The optical instrument of claim 20, further comprisingan excitation filter disposed along an excitation path between theexcitation light source and the beam splitter, the excitation filtercomprising a multiple bandpass filter and configured to transmit lightwithin the different excitation colors.
 22. The instrument of claim 20,further comprising a plurality of spaced-apart reaction regions disposedwithin the region of the apparatus, at least some of the reactionregions comprising at least one respective sample including afluorescent compound configured to produce an emission beam in responseto at least one of the excitation beams.
 23. The instrument of claim 20,wherein the beam splitter is a dichroic beam splitter.
 24. Theinstrument of claim 20, wherein the at least two light elementscomprises at least two light emitting diodes (“LEDs”).
 25. Theinstrument of claim 20, wherein the detector is a single array detectorconfigured to receive emission beams from more than one of the reactionregions.
 26. An optical instrument, comprising: an apparatus having aregion configured to hold a two-dimensional array of spaced-apartreaction regions; an excitation light source comprising a first lightelement configured to provide a first excitation beam having firstexcitation wavelength range and a second light element configured toprovide a second excitation beam having second excitation wavelengthrange that is different from the first excitation wavelength range; abeam splitter comprising a dichroic beam splitter having two passbands,the beam splitter configured to reflect or transmit the excitation beamsto the region of the apparatus to simultaneously illuminate more thanone of reaction regions so as to produce emission beams comprising atleast two different emission wavelength ranges; a detector; an imagingsystem comprising a lens configured to direct the excitation beams tosimultaneously illuminate the two-dimensional array of spaced-apartreaction regions and to direct the emission beams onto the detector toproduce two-dimensional images of the spaced-apart reaction regions;wherein the beam splitter is configured to reflect light from each ofthe light elements and to transmit light from the emission beams throughthe beam splitter and to the detector.